Production of recombinant human dornase alfa in Pichia pastoris

Dornase alfa (rhDNAseI) is one of the numerous nucleases common substance in nature that catalyze the hydrolysis of extracellular DNA. Dornase alfa that primary role is to cleaves nucleic acids and secreted by the pancreas and salivary glands. Dornase alfa was first cloned from the human pancreas in the late 1980s. Recombinant Dornase alfa, which selectively cleaves DNA, is used in the treatment of cystic fibrosis (CF) disease and shows its mechanism of action in the form of reducing viscosity in the lungs by hydrolyzing DNA contained in viscous mucus. In this study, a strain was developed to produces the human Dornase alfa enzyme recombinantly under the control of the ADH2SNT5 promoter which is ethanol-inducible and has a strong expression level in Pichia pastoris yeast. Dornase alfa production potential of the created-strain was studied at laboratory scale and was carried out scale-up studies to adapt it to industrial production. In addition, the expression of the Dornase alfa enzyme was carried out in the P. pastoris expression system for the first time in this study. In the first part of the study, the gene region encoding the Dornase alfa enzyme was cloned to the expression vector containing the synthetic ADH2SNT5 promoter region and pADH2SNT5αDornase alfa expression vector was obtained. All the production of Dornase alfa carried out as extracellular secretion in this study so P. pastoris MK115(PDI) containing 4 copies of the PDI chaperone protein was used as the host cell. Clones containing different plasmid copies were obtained from plate containing different zeocin ( 100-1000 µg/mL). Dornase alfa expression was performed in BMEY medium as erlenmayer scale with clones containing different copy numbers of Dornase alfa gene under standard production conditions (temperature of 28 ˚C and pH 6.0, 225 rpm). At the end of expression studies, pADH2SNT5αDornase alfa (MK115 PDI) #10 containing the clone single-copy Dornase alfa gene was selected as the production strain with the highest protein production. Later, different temperature and pH (20-28 ˚C and 3-7 pH range) experiments were carried out in order to increase the production amount at erlenmeyer scale. End of 96 hours of the production at erlenmayer scale, Dornase alfa enzyme activity tests were performed with the supernatant samples obtained from all the tested production conditions so production condition was determined pH 7.0 and the production temperature 24 ˚C according to the achieved highest Dornase alfa activity. In the last part of the study, some determined parameters (temperature 24 ˚C and pH 7.0) at the erlenmeyer scale were used to produce Dornase alfa in fermenter scale in minimal (FM22) and rich (BMGY) media by the created clone and the purification of histidine-labeled enzyme was performed by Fast Protein Liquid Chromatography (FPLC). The enzyme activity values after purification were determined 3525.42 U/ml (13559.32 U/mg) as a result of bioreactor production in FM22 medium while it was determined as 3325.42 U/ml (15254.23 U/mg) as a result of bioreactor production in BMGY medium. The main target of the study is to investigate potential of developing a product equivalent to the commercially available Dornase alfa enzyme. In this context with this study and future characterization studies, using the yeast cell platform instead of the mammalian expression system can create the potential for a higher yield product or equivalent to commercial product. KEYWORDS: Dornase alfa, fermentation, cystic fibrosis (CF), Pichia pastoris, production of recombinant Dornase alfa, rhDNAseI, ADH2SNT5 promoter